The field of the invention is the use of insulin-like growth factor I to affect vertebrate muscle mass and strength.
One of the primary consequences of aging leading to significantly impaired function in the elderly population is the loss of skeletal muscle strength and mass (Lamberts et al., 1997, Science 278:419-424). Both muscle mass and strength decrease by up to one third in humans between the ages of 30 and 80 years (Tzankoff et al., 1977, J. Appl. Physiol. 43:1001-1006). In addition, selective loss of the fastest, most powerful muscle fiber types (type IIb fibers) has been documented (Grimby et al., 1982, Acta Physiol. Scand. 115:125-134). Similar age-related muscle alterations have been observed in rats and mice, indicating that the trend is maintained in other mammalian species (Larrson and Edstrom, 1986, J. Neurol. Sci. 76:69-89; Brooks and Faulkner, 1988, J. Physiol. 404:71-82).
The loss of muscle mass and strength seen in aging mammals has given impetus to the search for compounds which may slow or reverse the process. One such compound is insulin-like growth factor I (IGF-I), which is a peptide growth factor that is structurally related to proinsulin (Cohick et al., 1993, Ann. Rev. Physiol. 55:131-153). IGF-I is critical in mediating the growth of muscle and other tissues (Florini et al., 1996, Endocrine Reviews 17:481-517). Systemic administration of IGF-I results in increased muscle protein content and reduced protein degradation (Zdanowicz et al., 1995, Endocrinology 136:4880-4886). Moreover, over-expression of IGF-I has been correlated with muscle hypertrophy in transgenic mouse lines (Coleman et al., 1995, J. Biol. Chem. 270:12109-12116). Further, addition of IGF-I to cultures of differentiating myocytes has been shown to result in hypertrophied myotubes (Florini et al., 1989, Am. J. Physiol. 256:C701-711).
Previous studies have also suggested that there is a correlation between aging-related loss of muscle mass and strength and decreased levels of IGF-I. Lamberts et a. (1997, Science 278:419-424), demonstrated that with aging, there is a decrease in the production and activity of the growth hormone/IGF-I axis which leads to an increase in catabolic processes as exhibited by the age-related loss of muscle mass and strength. The prevention of muscle mass loss has been achieved in healthy individuals by growth hormone administration, and is associated with increases in IGF-I levels (Papadakis et al., 1996, Ann. Intern. Med. 124:708-716).
The above-stated observations may also have important implications with regard to various muscular diseases. For example, aging-related changes in skeletal muscle mirror the early functional changes observed in muscular dystrophy, albeit on a much slower time scale. Just as in the muscles of old mice, the muscles of the mouse model of Duchenne muscular dystrophy (the mdx mouse) exhibit both decreased force per cross-sectional area and preferential loss of type IIb fibers (Petrof et al., 1993, Am. J. Physiol. 265 (Cell Physiol. 34):C834-C841). These data suggest that IGF-I expression in dystrophic muscle, while not addressing the primary defect that results in increased susceptibility to injury (Petrof et al., 1993, Proc. Natl. Acad. Sci. USA 90:3710-3714), may increase the rate of regeneration of the muscle and thereby tend to preserve muscle function.
Although the previous studies suggested that IGF-I may be useful in preserving muscle mass, these studies failed to demonstrate functional hypertrophy. That is, although there was an increase in muscle mass (i.e., hypertrophy), there was no concomitant increase in specific muscle strength (defined as force per cross-sectional area). For example, Coleman et al. (1995, J. Biol. Chem. 270:12109-12116), demonstrated that the hypertrophic muscles of transgenic mice did not exhibit enhanced strength compared with muscle obtained from nontransgenic, but otherwise identical animals. Indeed, the hypertrophied muscles of transgenic mice, although larger, were actually weaker than their non-transgenic counterparts (Leferovich et al, 1995, J. Neurosci. 14:596-603). Thus, while there are clear indications that IGF-I may be involved in maintaining muscle mass in elderly individuals, these studies did not suggest that muscle function, as measured by strength, may be preserved by the administration of IGF-I.
To date, the only means for stimulating muscle hypertrophy involve the use of steroids which have deleterious side effects. Systemic administration of IGF-I or growth hormone has proved ineffective ans also may have deleterious side effects. Thus, there is a long-felt and unfilled need for the development of compositions and procedures for promoting growth of adult muscle, which growth also serves to enhance the overall strength of the muscle. The present invention satisfies this need.
The invention relates to a method of increasing vertebrate muscle mass and muscle strength. The method comprises administering a muscle enhancing dose of an isolated nucleic acid encoding Insulin-like Growth Factor I (IGF-I), or a modification or biologically active portion thereof, intramuscularly into a vertebrate, wherein the isolated nucleic acid is expressed in muscle cells, thereby increasing the muscle mass and the muscle strength in the muscle of the vertebrate.
In one aspect, the vertebrate is selected from a group consisting of rat, mouse, cat, dog, horse, cow, pig, sheep, goat, fish, bird, and human.
In a preferred embodiment, the vertebrate is a human.
In another preferred embodiment, the IGF-I is of the same species as the vertebrate.
In another aspect, the isolated nucleic acid is contained within a virus vector.
In yet another aspect, the muscle enhancing dose ranges from between about 1010 to about 1012 recombinant virus vector particles per gram of muscle.
In yet another aspect; the method further comprises administering to the vertebrate fibroblast growth factor or neurotropin.
The invention also relates to an isolated nucleic acid comprising a vertebrate IGF-I coding region, or a modification or portion thereof, operably linked to a muscle specific promoter/regulatory region, wherein the IGF-I coding region is flanked on the 5xe2x80x2 side by an SV40 intron sequence and wherein the IGF-I coding region is flanked on the 3xe2x80x2 end by an SV40 polyadenylation signal sequence.
In one aspect, the muscle specific promoter/regulatory region is selected from a group consisting of the myosin light chain 1/3 promoter/enhancer, the skeletal xcex1-actin promoter, the muscle creatine kinase promoter/enhancer and a muscle specific troponin promoter.
In a preferred embodiment, the muscle specific troponin promoter is the fast troponin C promoter/enhancer.
In another preferred embodiment, the muscle specific promoter/regulatory region is the myosin light chain 1/3 promoter/enhancer.
In another aspect, the muscle specific promoter/regulatory region further comprises an enhancer element operably linked to the IGF-I coding region.
In a preferred embodiment, the enhancer is the myosin light chain 1/3 enhancer.
Also included in the invention is a composition comprising a recombinant virus vector comprising an isolated nucleic acid comprising a vertebrate IGF-I coding region, or a modification or portion thereof, operably linked to a muscle specific promoter/regulatory region, wherein said IGF-I coding region is flanked on the 5xe2x80x2 side by an SV40 intron sequence and wherein said IGF-I coding region is flanked on the 3xe2x80x2 end by an SV40 polyadenylation signal sequence. The muscle specific promoter/regulatory region further comprises an enhancer element operably linked to the IGF-I coding region and the enhancer is the myosin light chain 1/3 enhancer.
In one aspect, the recombinant virus vector is selected from the group consisting of an adeno-associated virus, an adenovirus and a herpes simplex virus.
In a preferred embodiment, the recombinant virus vector is an adeno-associated virus.
Also included in the invention is a cell comprising an isolated nucleic acid comprising a vertebrate IGF-I coding region, or a modification or portion thereof, operably linked to a muscle specific promoter/regulatory region, wherein said IGF-I coding region is flanked on the 5xe2x80x2 side by an SV40intron sequence and wherein said IGF-I coding region is flanked on the 3xe2x80x2 end by an SV40 polyadenylation signal sequence.
The invention further includes a cell comprising a recombinant virus vector comprising an isolated nucleic acid comprising a vertebrate IGF-I coding region, or a modification or portion thereof, operably linked to a muscle specific promoter/regulatory region, wherein said IGF-I coding region is flanked on the 5xe2x80x2 side by an SV40 intron sequence and wherein said IGF-I coding region is flanked on the 3xe2x80x2 end by an SV40 polyadenylation signal sequence. The muscle specific promoter/regulatory region further comprises an enhancer element operably linked to the IGF-I coding region and the enhancer is the myosin light chain 1/3 enhancer.
The invention additionally includes a kit for increasing muscle mass and muscle strength in a vertebrate. The kit comprises a muscle enhancing dose of an isolated nucleic acid comprising a vertebrate IGF-I coding region, or a modification or portion thereof, operably linked to a muscle specific promoter/regulatory region, wherein said IGF-I coding region is flanked on the 5xe2x80x2 side by an SV40 intron sequence and wherein said IGF-I coding region is flanked on the 3xe2x80x2 end by an SV40 polyadenylation signal sequence, wherein the isolated nucleic acid is expressed in vertebrate muscle cells, and wherein the kit further comprises an applicator for delivering the muscle enhancing dose, and instructions for the use of the kit.
Also included in the invention is a non-human transgenic vertebrate animal comprising an isolated nucleic acid encoding IGF-I, or a modification or biologically active portion thereof.
In one aspect, the IGF-I is operably linked to a muscle specific promoter/regulatory sequence at the 5xe2x80x2 end of the IGF-I and a polyadenylation termination signal at the 3xe2x80x2 end of the IGF-I.
In another aspect, the muscle specific promoter/regulatory sequence is selected from the group consisting of the myosin light chain 1/3 promoter/enhancer, the skeletal xcex1-actin promoter, the muscle creatine kinase promoter/enhancer and a muscle specific troponin promoter.
In yet another aspect, the non-human transgenic vertebrate animal is selected from the group consisting of rat, mouse, cat, dog, horse, cow, pig, sheep, goat, fish, bird, and human.